(i) Field of the Invention
The invention relates to modified starches having an elevated content of phosphate and an elevated content of amylose.
(ii) Description of the Related Art
In view of the increasing importance which is currently being attached to plant components as renewable sources of raw material, one of the tasks of biotechnological research is to endeavor to adapt these plant raw materials to the requirements of the processing industry. In addition to this, it is necessary to achieve a great diversity of substances in order to enable renewable raw materials to be used in as many areas of employment as possible.
While the polysaccharide starch is composed of chemically uniform basic units, i.e. the glucose molecules, it is a complex mixture of different molecular forms which exhibit differences with regard to the degree of polymerization and branching and consequently differ greatly from each other in their physicochemical properties. A distinction is made between amylose starch, an essentially unbranched polymer composed of alpha-1,4-glycosidically linked glucose units, and amylopectin starch, a branched polymer in which the branches are formed as a result of the appearance of additional alpha-1,6-glycosidic linkages. Another important difference between amylose and amylopectin lies in their molecular weights. While amylose, depending on the origin of the starch, has a molecular weight of 5×105-106 Da, the molecular weight of amylopectin is between 107 and 108 Da. The two macromolecules can be differentiated by their molecular weight and their different physicochemical properties, something which can most readily be visualized by their different iodine-binding properties.
Amylose was regarded for a long time as being a linear polymer which consisted of alpha-1,4-glycosidically linked alpha-D-glucose monomers. However, more recent studies have demonstrated the presence of alpha-1,6-glycosidic branching points (approx. 0.1%) (Hizukuri and Takagi, Carbohydr. Res. 134, (1984), 1-10; Takeda et al., Carbohydr. Res. 132, (1984), 83-92).
Different methods are available for determining the amylose content. Some of these methods are based on the iodine binding ability of the amylose, which ability can be determined potentiometrically (Banks & Greenwood, in W. Banks & C. T. Greenwood, Starch and its components (pp. 51-66), Edinburgh, Edinburgh University Press), amperometrically (Larson et al., Analytical Chemistry 25(5), (1953), 802-804) or spectrophotometrically (Morrison & Laignelet, J. Cereal Sc. 1, (1983), 9-20). The amylose content can also be determined calorimetrically by means of DSC (differential scanning calorimetry) measurements (Kugimiya & Donovan, Journal of Food Science 46, (1981), 765-770; Sievert & Holm, Starch/Stärke 45 (4), (1993), 136-139). In addition, it is possible to determine the amylose content of native or debranched starch using SEC (size exclusion chromatography). This method has been recommended, in particular, for determining the amylose content of recombinantly modified starches (Gérard et al., Carbohydrate Polymers 44, (2001), 19-27).
The functional properties, such as the solubility, the retrogradation behavior, the ability to bind water, the film-forming properties, the viscosity, the pasting properties, the freeze/thaw stability, the acid stability, the gel strength and the grain size of starches are influenced, inter alia, by the amylose/amylopectin ratio, the molecular weight, the pattern of side chain distribution, the content of ions, the content of lipid and protein, the mean starch grain size, the starch grain morphology, etc. The functional properties of starch are also influenced by the content of phosphate, i.e. a non-carbon component of starch. In this connection, a distinction is made between phosphate which is covalently bonded in the form of monoesters to the glucose molecules of the starch (termed starch phosphate here) and phosphate in the form of phospholipids which are associated with the starch.
The content of starch phosphate varies in dependence on the plant type. Thus, for example, certain corn mutants synthesize a starch having an elevated content of starch phosphate (waxy corn 0.002% and high-amylose corn 0.013%) whereas conventional corn types only exhibit traces of starch phosphate. Small quantities of starch phosphate are also found in wheat (0.001%) whereas it has not been possible to detect any starch phosphate in oats and sorghum. Less starch phosphate has also been found in rice mutants (waxy rice 0.003%) than in conventional rice types (0.013%). Significant quantities of starch phosphate have been detected in plants, such as tapioca (0.008%), sweet potato (0.011%), arrowroot (0.021%) and potato (0.089%), which synthesize tuber storage starch or root storage starch. The percentage values for the starch phosphate content which are cited above are in each case based on the dry weight of the starch and were determined by Jane et al. (1996, Cereal Foods World 41 (11), 827-832). In general, the distribution of the phosphate in (native) starch which is synthesized by plants is characterized by from about 30% to 40% of the phosphate residues being covalently bonded in the C3 position, and from about 60% to 70% of the phosphate residues being covalently bonded in the C6 position, of the glucose molecules (Blennow et al., 2000, Int. J. of Biological Macromolecules 27, 211-218). By contrast, chemically phosphorylated starches additionally possess phosphate residues which are covalently bonded in the C2 position of the glucose molecules since the chemical reaction proceeds in a randomly directed manner.
Kossmann and Lloyd (2000, Critical Reviews in Plant Sciences 19(3), 171-126) provide a review of native starches which are isolated from different plant species in which enzymes involved in starch biosynthesis are reduced.
Plants in which the activity of an SSIII protein (Abel et al., 1996, The Plant Journal 10(6), 9891-991; Lloyd et al., 1999, Biochemical Journal 338, 515-521) or the activity of a BEI protein (Kossmann et al., 1991, Mol Gen Genet. 230, 39-44; Safford et al., 1998, Carbohydrate Polymers 35, 155-168) or the activity of a BEII protein (Jobling et al., 1999, The Plant Journal 18), or the activity of a BEI and BEII protein (Schwall et al., 2000, Nature Biotechnology 18, 551-554; WO 96/34968, Hofvander et al., 2004, Plant Biotechnology 2, 311-321), or the activity of a BEI protein and of an SSIII (WO 00/08184) protein are reduced have thus far been described.
As compared with corresponding wild-type plants, starches which are isolated from plants in which the activity of an SSIII protein is reduced exhibit a relative shift of the side chains of the amylopectin from relatively long chains to short chains (Lloyd et al., 1999, Biochemical Journal 338, 515-521), a phosphate content which is elevated by 70%, no change in the amylose content (Abel et al., 1996, The Plant Journal 10(6), 9891-991) and a decrease in the final viscosity in the RVA analysis (Abel, 1995, Berlin Free University Dissertation). As compared with starches which are isolated from untransformed wild-type plants, these starches, which are also described in WO 00/08184, exhibit a phosphate content which is increased by 197%, an amylose content which is increased by 123% and a final viscosity in the RVA analysis which falls to 76% of the wild type. In addition, the gel strength of the starch concerned falls to 84% of the wild type.
In the Morrison & Laignelet (1983, J. Cereal Sc. 1, 9-20) spectrophotometric analysis, starches which are isolated from plants which exhibit a reduced activity of both a BEI protein and a BEII protein have an amylose content of from 77% to 89.1% (corresponds to at most 348% of the starch which is isolated from wild-type plants) and a phosphorus content of from 2400 μg/g of starch (corresponds to 77.4 μmol of phosphate/g starch) to 3000 μg/g of starch (corresponds to 96.8 μmol of phosphate/g starch). This gives a maximum increase of 613% as compared with starch which is isolated from corresponding wild-type plants. Starches containing more than 55% amylose no longer exhibit any pasting (Schwall et al., 2000, Nature Biotechnology 18, 551-554). Starches having lower amylose values (40.9%) exhibit a final viscosity value which is increased by 256%, after pasting in the RVA analysis, and exhibit a phosphorus content of 206 mg/100 g of starch (corresponds to 66.4 μmol of phosphate/g of starch). Higher phosphorus contents, e.g. 240 mg of phosphorus/100 g of starch (corresponds to 77.4 μmol of phosphate/g of starch; WO 9634968), are only achieved when the relevant starches also exhibit higher amylose values. Hofvander et al. (2004, Plant Biotechnology 2, 311-321) describe starches which are isolated from genetically modified potato plants having a phosphorus content of from 2400 to 3300 μg/g of starch (corresponds to from 77.4 to 106.4 μmol of phosphate/g of starch), with the starches exhibiting an amylose content (spectrophotometric determination of the iodine-binding ability) of from 47% to 86%.